We have previously demonstrates that the mTOR pathway might be associated with erythroid differentiation of K562 cells, in consideration of the fact that rapamycin (a strong and highly selective mTOR inhibitor) is able to induce erythroid differentiation of K562 cells. In this study we have analysed the effects of erythroid-inducer mithramycin (MTH) on two companion of mTOR, i.e. raptor and rictor. After K562 cells treatment with mithramycin, raptor and rictor content were analyzed by immunochemical analysis. The results obtained by quantitative RT-PCR clearly indicate that content of raptor mRNA, but not of rictor mRNA, decreases during erythroid induction of human leukemia K562 cells. These data have been confirmed using erythroid precursor cells (ErPC) from beta-thalassemic patients. In this case, ErPC were isolated from peripheral blood of beta-thalassemia patients, and then cultured with erythropoietin (EPO) in the presence of MTH. In a total of 15 cell cultures performed, we always observed a reduction of raptor mRNA accumulation and raptor production (when western blotting on protein extracts were performed). Furthermore, we have demonstrated that downstrean raptor/mTORC1 dependent biological activities were modulated following MTH treatment, including inhibition of phosphorilated p70S6K, of ribosomal S6 protein phosphorilation, increase of HIF transcription factor activity and increase of microRNA 210 content. This is particularly interesting, since this miR is able to target the 3’-UTR sequence of raptor mRNA, thereby participating in maintaining its down-regulation.
Mithramycin-mediated induction of K562 cell erythroid differentiation is associated with inhibition of the mTOR-CI patway
BIANCHI, Nicoletta;FINOTTI, Alessia;ZUCCATO, Cristina;FABBRI, Enrica;BORGATTI, Monica;GAMBARI, Roberto
2010
Abstract
We have previously demonstrates that the mTOR pathway might be associated with erythroid differentiation of K562 cells, in consideration of the fact that rapamycin (a strong and highly selective mTOR inhibitor) is able to induce erythroid differentiation of K562 cells. In this study we have analysed the effects of erythroid-inducer mithramycin (MTH) on two companion of mTOR, i.e. raptor and rictor. After K562 cells treatment with mithramycin, raptor and rictor content were analyzed by immunochemical analysis. The results obtained by quantitative RT-PCR clearly indicate that content of raptor mRNA, but not of rictor mRNA, decreases during erythroid induction of human leukemia K562 cells. These data have been confirmed using erythroid precursor cells (ErPC) from beta-thalassemic patients. In this case, ErPC were isolated from peripheral blood of beta-thalassemia patients, and then cultured with erythropoietin (EPO) in the presence of MTH. In a total of 15 cell cultures performed, we always observed a reduction of raptor mRNA accumulation and raptor production (when western blotting on protein extracts were performed). Furthermore, we have demonstrated that downstrean raptor/mTORC1 dependent biological activities were modulated following MTH treatment, including inhibition of phosphorilated p70S6K, of ribosomal S6 protein phosphorilation, increase of HIF transcription factor activity and increase of microRNA 210 content. This is particularly interesting, since this miR is able to target the 3’-UTR sequence of raptor mRNA, thereby participating in maintaining its down-regulation.I documenti in SFERA sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.